EP3225309A1 - Membranes for analysing microfluidic devices and manufacturing method - Google Patents
Membranes for analysing microfluidic devices and manufacturing method Download PDFInfo
- Publication number
- EP3225309A1 EP3225309A1 EP16163217.9A EP16163217A EP3225309A1 EP 3225309 A1 EP3225309 A1 EP 3225309A1 EP 16163217 A EP16163217 A EP 16163217A EP 3225309 A1 EP3225309 A1 EP 3225309A1
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/069—Absorbents; Gels to retain a fluid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
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- B01L2300/126—Paper
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
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- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
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Definitions
- the present invention relates to the general field of microfluidics. It relates more particularly to microfluidic devices for diagnostic purposes, as well as the manufacturing processes of such devices.
- microfluidic sensors these devices not only make it possible to analyze small volumes of liquid samples, they also make it possible to undertake, on a single platform, a plurality of detection or even quantification tests, targeted analytes and / or pathogens by simple and fast manipulation.
- the present invention is more specifically concerned with disposable microfluidic devices, of the type of those which comprise an analysis membrane made of a porous material and operating according to a so-called fluid lateral movement principle, in this case liquids.
- said analysis membrane has long been made from a sheet of cellulose fiber or nitrocellulose, the designation " ⁇ PAD" (for " microfluidic paper-based analytical devices” , in English) has been widely adopted to refer to this technology.
- ⁇ PAD for " microfluidic paper-based analytical devices” , in English
- Other porous, hydrophilic and absorbent materials as long as they can allow lateral movement of the liquids by capillarity, have also been envisaged and then improved in order to be able to act as analysis membranes for the fabrication of these microfluidic devices. These are for example materials made from nitrocellulose fibers, cellulose acetate, fabrics, porous films of polymers.
- the general principle of operation of this type of microfluidic device is essentially based on an analysis membrane made up / fabricated from a sheet of porous, hydrophilic and absorbent material, on which and in the bulk of which is formed a hydrophilic network formed areas of interest and channels.
- a liquid to be analyzed for example a liquid biological sample
- a series of qualitative and / or quantitative analyzes of its constituents can progress by simple capillarity and be submitted an analysis or a series of qualitative and / or quantitative analyzes of its constituents (immunodetection, molecular detection, affinity test, ligand-receptor coupling test, pH evaluation ).
- a porous substrate sheet is first impregnated with a negative photoresist (for example, photolithographic resin).
- a negative photoresist for example, photolithographic resin
- SU-8 2010 by immersion in a solubilized resin solution in a solvent. After removal of the excess resin, on one of its faces, the substrate sheet is coated with a mask material opaque to light waves.
- This mask is provided with perforated parts (or simply transparent to ultraviolet) and ultraviolet opaque parts which respectively produce the hydrophobic parts and the hydrophilic parts to be transferred onto the substrate.
- the assembly is then subjected to ultraviolet irradiation, at wavelengths capable of causing photopolymerization of the negative resin at the exposed areas.
- UV irradiation at wavelengths capable of causing photopolymerization of the negative resin at the exposed areas.
- hydrophilic zones delimited by impermeable borders are then drawn.
- An optional additional heat treatment makes it possible to completely evaporate the initial solvent and perfect the crosslinking.
- a substrate sheet is first impregnated with a conventional resin by immersion in a solution of resin solubilized with volatile solvent. The substrate sheet is then placed in a heated and ventilated chamber in order to evaporate the solvent and cause the resin to crosslink.
- the substrate sheet thus made entirely hydrophobic, is coated on its upper face with an etching mask.
- This mask has perforated parts and non-perforated parts, which respectively reproduce the hydrophilic parts and impermeable edges to be transferred to the substrate.
- a plasma or corona treatment is applied to the assembly and makes it possible to remove the resin present on the surface of the substrate, at the level of the exposed zones. At these exposed areas, the upper layers of the substrate find wettability and hydrophilic qualities.
- the method described in WO 2008/049083 consists in using an ink-stamp whose print head is specifically shaped to stamp a pattern with well-defined outlines.
- the ink-pad printing head is coated with hot, liquid wax and then applied to the porous substrate.
- the liquid wax deposited on the surface deeply impregnates the porous substrate. By hardening, it forms impermeable edges in the thickness of the substrate, which delimit hydrophilic zones.
- this method does not make it possible to produce graphically complex patterns.
- the final rendering is generally imperfect.
- the desired pattern is thus first designed by computer. Then, using a solid ink printer, it is printed with the desired image resolution on one side of a porous substrate sheet. With the printed side facing up, the substrate sheet is coated on a hot plate heated to a temperature of the order of 150 ° C, for about 2 minutes. Under the effect of heat, the wax contained in the printed ink melts and, by gravity, penetrates and impregnates the substrate in its thickness. By cooling, the wax resolidifies and forms, in the thickness of the substrate, impermeable edges defining hydrophilic zones, like the pattern initially designed by computer.
- the solid ink printers currently available commercially are unfortunately suitable only for printing on printing media (paper, transparent, cardboard) of standard sizes, well defined dimensions and whose thicknesses hardly exceed 250 microns.
- the thickness of the print medium is a limiting parameter which is however not only related to the computer hardware currently available, it is also related to the method itself.
- image resolution and impermeability of the borders formed the quality of the result decreases progressively with the increase of the thickness of the substrate.
- the thicker and more porous a printing substrate the more quantity / density of wax needed to form the impermeable edges will be important.
- the greater the quantity / density of wax required the more the lateral diffusion of the melted wax will be marked and deleterious for the fineness of the borders of the pattern initially designed in computer science.
- WO 2010/102294 describes an exemplary embodiment in which a XEROX ® Phaser 8560N printer is used to print on Whatman TM no. 1.
- This filter paper used as a porous substrate sheet has a cellulose fiber composition and a thickness of the order of 180 ⁇ m. Impermeable functional borders could be obtained by printing a 300 ⁇ m wax pattern. thickness (lateral); the impermeable edges thus formed were 850 ⁇ 50 ⁇ m thick. With wax plots with a thickness of less than 300 ⁇ m, it has not been possible to obtain waterproof borders that are functional.
- the way in which the printer is used in this method induces another limiting parameter, that of the constituent material of the porous substrate sheet to be printed. Indeed, it must be able to give the substrate sheet mechanical strength and tensile strength (or tear resistance) sufficient to allow it to be conveyed through the printer, and to be able to withstand all the constraints mechanical and thermal forces inflicted during the printing process.
- the porous substrate sheets made of flexible material, such as a fabric or a wadding, or a material of high friability, for example a fiberglass filter paper, can not be transformed / reworked by this method.
- the object of the present invention is to propose a process for manufacturing microfluidic devices, more specifically a process for making the analysis membrane part, which does not suffer from the abovementioned disadvantages. More specifically, it aims to improve the method described in WO 2010/102294 .
- An object of the present invention is therefore to provide a method of manufacturing analysis membranes for microfluidic devices whose implementation is not limited to the processing of sheets of cellulose fiber or nitrocellulose, but also to made sheets. in other porous materials, as well as substrate sheets of relatively large thickness, that is to say with a thickness of at least 200 ⁇ m, in particular between 200 ⁇ m and 1000 ⁇ m. .mu.m.
- Another objective of the present invention is also to be able to propose a method that is compatible with the constraints of an industrial operation of a single-use diagnostic device, particularly in terms of production cost and profitability.
- the present invention aims at proposing new analysis membranes whose originality lies not only in their design but also in their structure and / or the nature of the substrates used, as well as in the analytical performance that can be provided.
- the present invention therefore relates, firstly, to a method of manufacturing a microfluidic device analysis membrane; said microfluidic membrane being formed from a porous substrate sheet, in the thickness of which solid wax forms impermeable edges defining hydrophilic zones. These impermeable edges thus describe, through said substrate sheet, a pattern drawn with wax.
- said substrate sheet thus transformed is re-cut to the final dimensions of the desired analysis membrane.
- the lower face is covered with a sealing layer, for example, by spraying or by applying a resin composition or a wax, or by laminating an impermeable plastic film, for example of the poly (ethylene terephthalate) or PET type.
- this sealing layer may also have the advantage of stiffening and strengthening the structure of said analysis membrane.
- the term "sheet” is used in a very general and broad sense to designate a part, a piece of substrate whose thickness corresponds to an insignificant value with respect to its surface. In this context, this term is used here to designate a sheet in the strict sense, a ribbon or a disc.
- the term “film” will be used hereinafter to denote a piece, a piece of material whose thickness is significantly less than that of a sheet taken in the sense of the present invention.
- the term "waxes” denotes lipophilic substances, solid at room temperature (25 ° C.) and having a melting point of between 45 ° C. and 120 ° C. Just above the melting point, the waxes are liquid and have a particularly low viscosity. They can be of animal origin (for example, beeswax, Chinese wax, lanolin ...), vegetable (for example, rice wax, mimosa, candellila wax, carnauba, the wax of Japan, the vegetaline %), mineral (for example, paraffin, ozokerite %) or synthetic (for example, stearin, ethylene polymers, fatty amines, polyethylene glycol or PEG, ).
- the wax deposited on the underside of the substrate sheet gradually permeates the substrate in all directions, including upwards.
- the vertical path of the wax is reduced. In doing so, the deep impregnation is favored and the migration time is shortened. Less freedom is left to the wax to be able to diffuse in one direction radial / lateral; the smoothness of the initial wax pattern is affected less significantly.
- the thickness of said substrate sheet is between 200 ⁇ m and 1000 ⁇ m, preferably between about 250 ⁇ m and about 850 ⁇ m.
- said heat treatment is carried out inside a thermo-controlled heating chamber.
- said mechanical treatment it is implemented by mechanical means able to apply a pressure or a compressive force sufficient to compress the thickness of said substrate sheet, at least at the intermediate units, installed at the same time. interior of said thermo-controlled enclosure.
- these mechanical means may consist of one or more plates, having a certain mass, which is applied to the substrate sheet. It may also be a vise device, adapted to maintain, between its jaws, the substrate sheet in a position at least substantially horizontal, and to compress the thickness.
- thermo-controlled heating compression device provided with two horizontal heating compression plates.
- This may include a hot press (or hot press).
- thermo-controlled compression device a layer of flexible and elastic material, at least substantially flat, is interposed between each of the faces of said substrate sheet and the heating compression plates. Said layer of flexible and elastic material then serves as a thermal and mechanical buffer; On the one hand, it makes it possible to slow the transmission of heat substrate and, on the other hand, more evenly distribute the heat and pressure over the entire substrate sheet.
- a thermal and mechanical buffer element one can for example use a piece of rubber or any other similar polymeric material, a piece of tissue, a piece of cork.
- Cooling can be done passively, stopping heating and / or removing and moving the substrate sheet away from any heat source.
- mechanical expansion can be achieved simply by quickly removing the mechanical stresses applied or by gradually reducing their intensity.
- the chronological order in which thermal expansion and mechanical expansion are triggered is not very important.
- the two detents are triggered simultaneously or one after the other.
- any suitable techniques known to those skilled in the art can be used.
- 3D printing can be used.
- the intermediate patterns are traced directly on the substrate sheet by deposition of wax.
- the intermediate pattern reproduces identically the desired final pattern.
- the intermediate pattern appears as an inverted image of the desired final pattern.
- the two intermediate units are positioned at least substantially opposite one another.
- the waxed intermediate units are affixed to each of the faces of the porous substrate sheet with a wax transfer technique.
- the intermediate patterns are first printed on a transfer film.
- the intermediate pattern intended to coat the underside of the substrate sheet appears identical to the final pattern to be produced, whereas the intermediate pattern intended to coat the upper face of the substrate sheet appears as an image. inverted of the first intermediate pattern.
- a solid ink printer can advantageously be used to print the intermediate patterns.
- the amount (per unit area) of wax deposited on the transfer film depends on the print quality chosen, the inks used - more precisely the wax concentration present in these compositions. ink-, and the intensity of the coloring of the patterns to be printed.
- transfer film usable for the implementation of the method according to the invention various materials are possible, since they are printable. This may include a printable plastic sheet (transparent or opaque), a sheet of printing paper, a sheet of parchment paper.
- the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on two separate transfer films.
- the substrate sheet is then placed between the two transfer films, the wax patterns being pressed against the faces of the substrate sheet.
- the assembly is held together and is subjected to heat and mechanical treatments according to the invention.
- the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on one and the same transfer film.
- the two intermediate units are arranged side by side, symmetrically with respect to an axis.
- the transfer film is folded in half along said axis of symmetry with the printed side turned inward.
- the two intermediate patterns are thus superimposed.
- the substrate sheet is inserted inside the folded transfer film between the two printed intermediate patterns. The assembly is held together and is subjected to said thermal and mechanical treatments according to the invention.
- the transfer film is a paper sheet of cellulose fiber. Slightly absorbent, this film makes it possible, during the thermal and mechanical treatments according to the invention, to capture at least a portion of the molten wax remaining on the surface of the substrate before it slinks / flows on the sides.
- the method according to the invention can be implemented from a wide range of porous substrate sheets, in particular from a fibrous composition chosen from: cellulose, nitrocellulose, cotton, linter, glass, silk fiber , viscose, polypropylene, polyester, polyamide (Nylon ® ), poly (lactic acid) or PLA.
- Said fibers may optionally be functionalized and / or loaded and / or doped with additives (for example, talc, diatomite, etc.).
- the process according to the invention is carried out starting from a porous substrate sheet of the same composition as a filtering medium made of cellulose fiber, nitrocellulose, cotton, linter, silk, viscose, polypropylene, polyester, preferably fiberglass.
- said method of manufacturing a microfluidic device analysis membrane is implemented from a porous fiberglass substrate sheet.
- said substrate sheet contains the composition of a fiberglass filter medium.
- Fiberglass filtering media are characterized by a great fragility in tearing and crumbling. This great structural fragility makes them incompatible with any direct printing by means of solid ink printers currently on the market. The mechanical constraints imposed by them are indeed considerably denaturing.
- the merit of the inventors in addition to having considered the use of a fiberglass filtering medium as a porous substrate for making analysis membranes of / or for microfluidic devices, it is the merit of the inventors to have succeeded in point a process capable of integrating into the thickness of this particular material, wax to form in fine impervious edges defining so-called hydrophilic zones.
- the proposed method allows the production, in the thickness of the relatively thick substrate sheets (between 200 microns and 1000 microns), curbs waxes perfectly impermeable to liquids and whose fineness (lateral thickness) can reach of the order of 800 microns.
- the present invention relates, secondly, a microfluidic device analysis membrane, capable of being manufactured by a method according to the invention.
- the said test membrane has a thickness of which solid wax forms impermeable (in this case, liquid impermeable) borders delimiting hydrophilic zones.
- the thickness of the analysis membrane is between 200 ⁇ m and 1000 ⁇ m, preferably between about 250 ⁇ m and about 850 ⁇ m.
- said analysis membrane comprises a face (in this case, a so-called lower face) covered with a sealing layer.
- a face in this case, a so-called lower face
- a sealing layer This can be obtained by spraying or by applying a resin composition or a wax, or by laminating a plastic film, for example of the PET type.
- the constitutive wax of said impermeable edges is chosen from an animal wax (for example, beeswax, Chinese wax, lanolin, etc.) and vegetable (for example, rice wax). , mimosa, candellila wax, carnauba wax, Japan wax, vegetaline ...), mineral (for example, paraffin, ozokerite %) and synthetic (for example, stearin, polymers ethylenic, fatty amines, polyethylene glycol or PEG .
- animal wax for example, beeswax, Chinese wax, lanolin, etc.
- vegetable for example, rice wax
- mineral for example, paraffin, ozokerite
- synthetic for example, stearin, polymers ethylenic, fatty amines, polyethylene glycol or PEG .
- the analysis membrane is made of cellulose fiber, nitrocellulose fiber or fiberglass, cotton, linter, cellulose wadding, silk fiber, viscose, polypropylene, of polyester. It may optionally be functionalized and / or charged and / or doped with additives (for example, talc, diatomite, etc.).
- the analysis membrane is made in a porous substrate whose composition resumes that of a filtering medium of cellulose fiber, nitrocellulose, cotton, linter, glass, silk, viscose , polypropylene, polyester, polyamide or PLA.
- the analysis membrane has a thickness of between 200 microns and 1000 microns, typically between about 250 microns and about 850 microns.
- the analysis membranes made of fiberglass according to the invention allow an analysis of the plasma directly from the whole blood.
- the blood deposited on the surface of the membrane is fractionated; cells and plasma are separated.
- the cells remain fixed in the deposition zone while the plasma will diffuse through the membrane, progressing along the hydrophilic channels by lateral diffusion.
- the analysis membranes are conventionally made of cellulose fiber or nitrocellulose. These analysis membranes alone are incapable of separating the plasma fraction from the cell fraction. Whole blood deposited on such analysis membranes coagulates rapidly; after the deposition of the blood sample on these supports, the cells block the plasma and prevent its lateral diffusion through the hydrophilic channels.
- the ⁇ PADs heretofore proposed comprise an assembly of an analysis membrane with a filtration system (for example, a fiberglass filtering medium) capable of implementing a fractionation of the blood. ; once separated from the cells that remain blocked at the level of the filtration system, the plasma diffuses through the analysis membrane where its components are then analyzed.
- ⁇ PAD comprising such an assembly between an analysis membrane and a filter medium is described by Songjaroen et al., 2012 (Lab Chip, 2012 (12) 3392-3398: "Blood separation on microfluidic paper-based analytical device” ).
- said analysis membrane in addition to impermeable edges drawn with wax and delimiting hydrophilic zones, also comprises, in its thickness, encrustations of wax, present at the level of at least one hydrophilic zone.
- encrustations do not form impermeable barriers to passage of liquids. Positioned at various hydrophilic zones, they are intended to disrupt and / or slow down the flow of liquid locally and / or to create well-defined reservoir and / or reaction zones. They can also define the contours of wax-free zones, thus very hydrophilic, which can make use of reservoirs or well-defined reaction zones.
- a first method consists of taking the previously described method to establish the impermeable edges of the analysis membrane. Localized and localized incrustations of wax are then obtained which extend through the entire thickness of said sheet. They thus form obstacles which the liquids must circumvent by the sides to be able to progress through the membrane of analysis.
- Another method is to repeat the process according to the invention but by modifying it somewhat to disperse in the thickness of the porous substrate a small amount of wax, insufficient to completely block the migration of liquids.
- the present invention also extends to a microfluidic device comprising at least one analysis membrane according to the invention.
- the present invention also relates to a method for manufacturing a microfluidic device analysis membrane, a microfluidic device analysis membrane and a microfluidic device characterized, in combination, by all or some of the features above or above. -after.
- the Figure 1 schematically illustrates the implementation of a method of manufacturing an analysis membrane 10, according to the invention, and by which a pattern drawn with wax is integrated into the thickness of a sheet of porous substrate 11; the contours of said pattern being intended to form, in the thickness of said substrate sheet 11, impermeable borders 12 defining hydrophilic zones 13 of determined configuration.
- the two intermediate units 12a and 12b are printed on one and the same transfer film 20.
- an image 12'a corresponding to the pattern to be integrated in the thickness of the substrate sheet 11 is created by computer and by means of a drawing software. This first image 12'a is duplicated in FIG. a symmetrical image 12'b.
- the two images 12'a and 12'b are printed on a transfer film 20, so as to form two intermediate units 12a and 12b arranged symmetrically with respect to an axis.
- This axis of symmetry S is also printed on the transfer film 20, as a visual cue.
- the printing is carried out with a XEROX ® ColorQube TM type printer, supplied with solid inks of reference XEROX ® 108R00931 (cyan color), 108R00932 (magenta color), 108R00933 (yellow color) and 108R00934 / 108R00935 ( black color).
- the transfer film 20 is an ordinary office paper sheet. The contours of the pattern are printed in quality / photo resolution in black ink.
- the transfer film 20 is folded in two along the axis of symmetry S, the intermediate units 12a and 12b turned inwards. The latter are thus superimposed on one another.
- a substrate sheet 11 is slid inside the folded transfer film 20, interposed between the two intermediate units 12a and 12b.
- the substrate sheet 11, sandwiched between the two flaps of the transfer film 20, is then pressed between two horizontal heating plates and two rubber parts 15 forming a thermal and mechanical buffer.
- the assembly is subjected to a pressure of the order of 1 kg / cm 2 and at a temperature of 120 ° C for about 3 minutes. During this process, the wax previously printed on the transfer film 20 is transferred onto both sides of the substrate sheet 11 and then impregnates the thickness.
- FIG. 2 is a photograph of a WHATMAN TM MF1 membrane after an unsuccessful direct print attempt. This membrane, previously taped on a sheet of standard size paper to facilitate its passage through the printer and to consolidate somewhat the structure, comes out of the printer completely deteriorated.
- the Figure 3 illustrates, in its part (A), a transfer film 20 on which a particular example of intermediate units 12a and 12b is printed.
- part (B) is exposed a photograph of an analysis membrane 10 (after use) formed from a fiberglass substrate sheet and using such a transfer film 20.
- the intermediate units 12a and 12b of generally rectilinear shape, have a flared upper part and a narrow lower part with an open end. Their contours are printed in black ink (reference XEROX ® 108R00934), in photo quality.
- This solid ink contains a wax concentration which has been found to be sufficient to achieve and obtain impermeable edges in the thickness of relatively thick substrate sheets, including WHATMAN TM VF2 membranes of 785 ⁇ m in thickness.
- the upper part of the intermediate units 12a and 12b is flared and is intended to form a hydrophilic zone, in this case a deposition zone 13a able to receive a liquid sample to be analyzed.
- a deposition zone 13a able to receive a liquid sample to be analyzed.
- the liquid sample will be able to migrate by capillarity towards the other hydrophilic zones of the analysis membrane, namely towards the lower part of the pattern.
- Narrower it forms a channel within which four zones of interest 13b, 13c, 13d and 13e are formed.
- the position of each of these areas of interest is specifically identified by the marking elements 14b, 14c, 14d and 14e, also drawn in colored solid ink.
- a low-wax color layer 14 covers the surface of the transfer film. 20.
- the low-wax layer 14 is obtained by printing, in solid ink, a light-colored layer (weakly intense). The intensity of the color of this layer is proportional to the amount of wax deposited on the transfer film; this color intensity is chosen so that, once in the thickness of the substrate sheet, the amount of wax thus transferred is insufficient to completely block the migration of liquids by capillarity.
- Areas of interest 13b, 13c, 13d and 13e were not modified by the wax. They retain the initial porosity and hydrophilicity of the substrate sheet used, and may be functionalized later.
- the various more or less hydrophilic zones will be able to be functionalized and / or loaded with reagents according to the desired analyzes and tests.
- the Figure 4 discloses the photograph of two analysis membranes made from a WHATMAN TM MF1 membrane-type substrate sheet (367 ⁇ m thick) and using a transfer film 20, as set forth in FIG. Figure 3 and as described in the previous example.
- the analysis membrane of the photograph (A) was manufactured using a method according to the invention and as illustrated in FIG. Figure 1 .
- the analysis membrane of the photograph (B) was made using the same method, but omitting the mechanical treatment (compression of the substrate sheet to about 1 kg / cm 2 ).
- the two analysis membranes are loaded with a colored solution. Unlike the analysis membrane of the photograph (A), with that of the photograph (B), large leaks of the colored solution are observed at the level of the deposition zone. The amount of wax transferred is significantly lower and does not allow to obtain pattern impermeable walls.
- the impermeability to liquids is obtained for a wax thickness printed at least equal to 260 ⁇ m, giving after transfer a wax barrier of the order of 830 ⁇ m. Below this thickness, the colorant is no longer contained in the pattern. circular.
- the tightness of the patterns is checked by means of a deposit of a colored solution in the flared portion of the pattern (deposition zone).
- the analysis membrane 10 has been functionalized for the detection of hepatitis B, by immunodetection of the HBs antigen contained in the blood.
- the zone 13d is functionalized by means of monoclonal antibodies anti-HBs, specific for the reaction; zone 13d forms the "spot test".
- the 13th zone is functionalized by means of anti-alkaline phosphatase monoclonal antibodies, specific for the detection conjugate; the 13th zone forms the "positive control spot”.
- the zone 13c is functionalized by means of non-specific antibodies of the reaction (for example, anti-rat antibodies); zone 13c forms the "negative control spot".
- the functionalization of these different zones by the antibodies is carried out by passive adsorption of the antibodies on the constituent fibers of the substrate.
- zone 13b is dedicated to storage of the second part of the conjugate complex (monoclonal antibodies anti-HBs labeled with biotin); zone 13b forms the "anti-HBs-biot Ac spot".
- the deposition zone 13a can also be used for storing the conjugate of the enzyme-linked immunosorbent reaction (streptavidin-alkaline phosphatase or STRE-PAL) in dried form.
- This conjugate will be resolubilized by the liquid phase of the sample to be analyzed.
- the analysis membrane 10 According to a second mode of application of the analysis membrane 10 previously described, it has been functionalized for the detection, in blood and plasma, of two proteins of the dengue virus: the NS1 protein and the domain III of the envelope protein of the virus (DomIII).
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Abstract
L'invention concerne un procédé de fabrication d'une membrane d'analyse (10) de dispositif microfluidique ; ladite membrane d'analyse étant formée à partir d'une feuille de substrat poreux (11) dans l'épaisseur de laquelle de la cire solide forme des bordures imperméables (12) délimitant des zones hydrophiles (13) et décrivant, à travers ladite feuille de substrat (11), un motif tracé à la cire ;
caractérisé en ce qu'il comprend les étapes suivantes :
- un motif intermédiaire (12a, 12b), façonné à la cire à l'image dudit motif tracé à la cire que formeront les bordures imperméables de la membrane d'analyse (11), est apposé sur chacune des faces d'une feuille de substrat poreux (11) de sorte que, de part et d'autre de l'épaisseur de ladite feuille de substrat (11), lesdits motifs intermédiaires (12a, 12b), mutuellement symétriques, se trouvent positionnés au moins sensiblement en regard l'un de l'autre ;
- maintenue au moins sensiblement à l'horizontale, ladite feuille de substrat (11) est soumise à un traitement thermique apte à provoquer une fusion au moins partielle de la cire constitutive des motifs intermédiaires (12a, 12b) apposés sur les faces de ladite feuille de substrat (11), et à un traitement mécanique apte à comprimer l'épaisseur de tout ou partie de ladite feuille de substrat (11);
- ladite feuille de substrat (11) est soumise à une phase de détente mécanique et thermique apte à permettre à ladite feuille de substrat (11) de reprendre au moins en partie son épaisseur initiale et à la cire de se resolidifier à l'intérieur de l'épaisseur de ladite feuille de substrat (11) ; et
en ce que l'épaisseur de ladite feuille de substrat (11) est comprise entre 200 µm et 1000 µm
The invention relates to a method for manufacturing a microfluidic device analysis membrane (10); said analysis membrane being formed from a porous substrate sheet (11) in the thickness of which solid wax forms impermeable borders (12) defining hydrophilic zones (13) and describing, through said sheet substrate (11), a pattern drawn with wax;
characterized in that it comprises the following steps:
- an intermediate pattern (12a, 12b), shaped with wax in the image of said wax pattern formed by the impermeable edges of the analysis membrane (11), is affixed to each of the faces of a sheet of a porous substrate (11) such that, on either side of the thickness of said substrate sheet (11), said mutually symmetrical intermediate units (12a, 12b) are positioned at least substantially opposite to each other; one of the other;
- Maintained at least substantially horizontally, said substrate sheet (11) is subjected to a heat treatment capable of causing at least partial melting of the constituent wax of the intermediate units (12a, 12b) affixed to the faces of said sheet substrate (11), and a mechanical treatment capable of compressing the thickness of all or part of said substrate sheet (11);
said substrate sheet (11) is subjected to a mechanical and thermal expansion phase able to allow said substrate sheet (11) to take up at least part of its initial thickness and the wax to resolidify inside the the thickness of said substrate sheet (11); and
in that the thickness of said substrate sheet (11) is between 200 μm and 1000 μm
Description
La présente invention a trait au domaine général de la microfluidique. Elle concerne plus particulièrement les dispositifs microfluidiques à visée diagnostique, ainsi que les procédés de fabrication de tels dispositifs.The present invention relates to the general field of microfluidics. It relates more particularly to microfluidic devices for diagnostic purposes, as well as the manufacturing processes of such devices.
La microfluidique peut être définie comme l'étude des phénomènes qui régissent le mouvement de petits volumes de fluide, notamment de liquide. Elle englobe le développement de systèmes et de dispositifs permettant de faire circuler et/ou de manipuler de petits volumes de fluides et ce, à des fins très diverses comme, par exemple :
- pour mélanger, éventuellement pour faire réagir, tout ou partie des constituants d'un ou de plusieurs fluides, entre eux et/ou avec des composés exogènes,
- pour séparer certains constituants d'un fluide, éventuellement en vue d'en analyser les propriétés chimiques et/ou physiques,
- pour détecter, voire pour doser, des agents cibles dont on suspecte la présence.
- for mixing, optionally to react, all or part of the constituents of one or more fluids, with each other and / or with exogenous compounds,
- to separate certain constituents of a fluid, possibly for the purpose of analyzing the chemical and / or physical properties thereof,
- to detect, or even to dose, target agents whose presence is suspected.
Dans le domaine de la clinique, les tests de diagnostic mettant en oeuvre des dispositifs microfluidiques ont connu une rapide évolution depuis ces deux-trois dernières décennies (
Aussi désignés par « capteurs microfluidiques », ces dispositifs rendent non seulement possible l'analyse de petits volumes d'échantillons liquides, ils permettent également d'entreprendre, sur une seule et même plateforme, une pluralité de tests de détection, voire de quantification, d'analytes et/ou d'agents pathogènes cibles et ce, par une manipulation simple et rapide.Also called "microfluidic sensors", these devices not only make it possible to analyze small volumes of liquid samples, they also make it possible to undertake, on a single platform, a plurality of detection or even quantification tests, targeted analytes and / or pathogens by simple and fast manipulation.
La présente invention s'intéresse plus précisément aux dispositifs microfluidiques à usage unique, du type de ceux qui comprennent une membrane d'analyse réalisée dans un matériau poreux et fonctionnant selon un principe dit de mouvement latéral des fluides, en l'occurrence des liquides. Parce que ladite membrane d'analyse a longtemps été réalisée à partir d'une feuille en fibre de cellulose ou de nitrocellulose, l'appellation «µPAD» (pour "microfluidic paper-based analytical devices", en anglosaxon) a été largement adoptée pour faire référence à cette technologie. D'autres matériaux poreux, hydrophiles et absorbants, dès lors qu'ils peuvent autoriser un mouvement latéral des liquides par capillarité, ont aussi été envisagés puis améliorés, pour pouvoir faire office de membranes d'analyse pour la confection de ces dispositifs microfluidiques. Il s'agit par exemple de matériaux réalisés à partir de fibres de nitrocellulose, d'acétate de cellulose, des tissus, des films poreux en polymères.The present invention is more specifically concerned with disposable microfluidic devices, of the type of those which comprise an analysis membrane made of a porous material and operating according to a so-called fluid lateral movement principle, in this case liquids. Because said analysis membrane has long been made from a sheet of cellulose fiber or nitrocellulose, the designation "ΜPAD" (for " microfluidic paper-based analytical devices" , in English) has been widely adopted to refer to this technology. Other porous, hydrophilic and absorbent materials, as long as they can allow lateral movement of the liquids by capillarity, have also been envisaged and then improved in order to be able to act as analysis membranes for the fabrication of these microfluidic devices. These are for example materials made from nitrocellulose fibers, cellulose acetate, fabrics, porous films of polymers.
Le principe général de fonctionnement de ce type de dispositif microfluidique repose essentiellement sur une membrane d'analyse confectionnée/façonnée à partir d'une feuille de matériau poreux, hydrophile et absorbant, sur laquelle et dans la masse de laquelle est ménagé un réseau hydrophile formé de zones d'intérêt et des canaux. A l'intérieur et au travers de ce réseau de zones d'intérêt et de canaux, un liquide à analyser (par exemple un échantillon biologique liquide), une fois déposé sur ladite membrane d'analyse, peut progresser par simple capillarité et être soumis à une analyse ou une série d'analyses qualitatives et/ou quantitatives de ses constituants (immunodétection, détection moléculaire, test d'affinité, test de couplage ligand-récepteur, évaluation du pH...).The general principle of operation of this type of microfluidic device is essentially based on an analysis membrane made up / fabricated from a sheet of porous, hydrophilic and absorbent material, on which and in the bulk of which is formed a hydrophilic network formed areas of interest and channels. Inside and through this network of areas of interest and channels, a liquid to be analyzed (for example a liquid biological sample), once deposited on said analysis membrane, can progress by simple capillarity and be submitted an analysis or a series of qualitative and / or quantitative analyzes of its constituents (immunodetection, molecular detection, affinity test, ligand-receptor coupling test, pH evaluation ...).
De manière très schématique, sur les membranes d'analyse des dispositifs microfluidiques, il peut être distingué :
- des zones dites zones de dépôt, au niveau desquelles les liquides à analyser sont déposés,
- des zones dites zones de réaction, au niveau desquelles ont lieu les réactions de détection et/ou de dosage,
- éventuellement, des zones mixtes qui font office simultanément de zones de dépôt et de zones de réaction,
- éventuellement, des zones dites zones réservoirs, au niveau desquelles des réactifs et/ou additifs nécessaires aux réactions à mener sont stockés, généralement sous forme sèche avant d'être réhydratés (ou solubilisés) et transférés vers des zones de réaction spécifiques,
- éventuellement, des zones de dépôt secondaires, qui seront alimentées en réactif(s) et/ou en additif(s), extemporanément et/ou au cours du processus d'analyse, et
- des structures formant canaux qui assurent une conduction fluidique entre les zones d'intérêt, et à travers lesquelles les liquides progressent par un simple phénomène de capillarité.
- areas called deposition zones, at which the liquid to be analyzed are deposited,
- zones called reaction zones, at which the detection and / or dosing reactions take place,
- possibly mixed zones which simultaneously act as deposition zones and reaction zones,
- optionally, so-called reservoir zones, at which reagents and / or additives necessary for the reactions to be carried out are stored, generally in dry form before being rehydrated (or solubilized) and transferred to specific reaction zones,
- optionally, secondary deposition zones, which will be fed with reagent (s) and / or additive (s), extemporaneously and / or during the analysis process, and
- channel structures that provide fluidic conduction between the areas of interest, and through which the liquids progress by a simple capillarity phenomenon.
S'agissant de la conception de tels dispositifs et plus particulièrement de la partie formant membrane d'analyse, plusieurs approches ont été proposées pour créer, à travers l'épaisseur d'une feuille de substrat poreux, de telles zones hydrophiles délimitées par des bordures imperméables.With regard to the design of such devices and more particularly of the analysis membrane part, several approaches have been proposed to create, through the thickness of a porous substrate sheet, such hydrophilic zones delimited by borders. waterproof.
Parmi les méthodes proposées, celle décrite par
Cette méthode, particulièrement consommatrice en résine, est très coûteuse à mettre en oeuvre, compte tenu du coût élevé des résines photosensibles.This method, particularly consuming resin, is very expensive to implement, given the high cost of photosensitive resins.
Une alternative à l'emploi de résines photosensibles est proposée par
Cette méthode est également très consommatrice en résine. Qui plus est, elle nécessite un équipement sophistiqué et coûteux, et dont l'investissement financier se justifie difficilement au regard du prix de vente envisageable pour des dispositifs de diagnostic à usage unique.This method is also very consuming in resin. What is more, it requires sophisticated and expensive equipment, whose financial investment is justified. with regard to the possible selling price for single-use diagnostic devices.
De mise en oeuvre plus rudimentaire, la méthode décrite dans
Utilisant également de la cire pour la conception des bordures imperméables délimitant des zone hydrophiles, la méthode décrite par
Dans cette méthode, le motif désiré est ainsi d'abord conçu par ordinateur. Puis, au moyen d'une imprimante à encre solide, celui-ci est imprimé avec la résolution d'image désirée sur une des faces d'une feuille de substrat poreux. Avec la face imprimée orientée vers le haut, la feuille de substrat est couchée sur une plaque chauffante portée à une température de l'ordre de 150°C, pendant environ 2 minutes. Sous l'effet de la chaleur, la cire contenue dans l'encre imprimée fond et, par gravité, pénètre et imprègne le substrat dans son épaisseur. En refroidissant, la cire se resolidifie et forme, dans l'épaisseur du substrat, des bordures imperméables délimitant des zones hydrophiles, à l'image du motif initialement conçu par ordinateur.In this method, the desired pattern is thus first designed by computer. Then, using a solid ink printer, it is printed with the desired image resolution on one side of a porous substrate sheet. With the printed side facing up, the substrate sheet is coated on a hot plate heated to a temperature of the order of 150 ° C, for about 2 minutes. Under the effect of heat, the wax contained in the printed ink melts and, by gravity, penetrates and impregnates the substrate in its thickness. By cooling, the wax resolidifies and forms, in the thickness of the substrate, impermeable edges defining hydrophilic zones, like the pattern initially designed by computer.
Les imprimantes à encre solide actuellement disponibles dans le commerce ne sont malheureusement adaptées que pour imprimer sur des support d'impression (papier, transparent, carton) de formats standards, aux dimensions biens définies et dont les épaisseurs ne dépassent guère 250 µm. L'épaisseur du support d'impression est un paramètre limitant qui n'est cependant pas uniquement lié au matériel informatique actuellement disponible, elle est également liée à la méthode elle-même. En termes de résolution d'image et d'imperméabilité des bordures formées, la qualité du résultat décroit progressivement avec l'augmentation de l'épaisseur du substrat. Plus un substrat d'impression est épais et poreux, plus la quantité/densité de cire nécessaire pour former les bordures imperméables sera importante. Egalement, plus la quantité/densité de cire nécessaire est importante et plus la diffusion latérale de la cire fondue sera marquée et délétère pour la finesse des bordures du motif initialement conçu en informatique.The solid ink printers currently available commercially are unfortunately suitable only for printing on printing media (paper, transparent, cardboard) of standard sizes, well defined dimensions and whose thicknesses hardly exceed 250 microns. The thickness of the print medium is a limiting parameter which is however not only related to the computer hardware currently available, it is also related to the method itself. In terms of image resolution and impermeability of the borders formed, the quality of the result decreases progressively with the increase of the thickness of the substrate. The thicker and more porous a printing substrate, the more quantity / density of wax needed to form the impermeable edges will be important. Also, the greater the quantity / density of wax required, the more the lateral diffusion of the melted wax will be marked and deleterious for the fineness of the borders of the pattern initially designed in computer science.
A titre indicatif,
La façon dont est utilisée l'imprimante dans cette méthode induit un autre paramètre limitant, celui du matériau constitutif de la feuille de substrat poreux à imprimer. En effet, celui-ci doit pouvoir conférer à la feuille de substrat une tenue mécanique et une résistance en traction (ou résistance à la déchirure) suffisantes pour lui permettre d'être convoyée à travers l'imprimante, et pour pouvoir supporter toutes les contraintes mécaniques et thermiques infligées pendant le processus d'impression. Les feuilles de substrat poreux réalisées en matériau souple, tel qu'un tissu ou une ouate, ou en un matériau de grande friabilité, par exemple un papier filtre en fibre de verre, ne peuvent être transformées/remaniées par cette méthode.The way in which the printer is used in this method induces another limiting parameter, that of the constituent material of the porous substrate sheet to be printed. Indeed, it must be able to give the substrate sheet mechanical strength and tensile strength (or tear resistance) sufficient to allow it to be conveyed through the printer, and to be able to withstand all the constraints mechanical and thermal forces inflicted during the printing process. The porous substrate sheets made of flexible material, such as a fabric or a wadding, or a material of high friability, for example a fiberglass filter paper, can not be transformed / reworked by this method.
La présente invention a pour objet de proposer un procédé de fabrication de dispositifs microfluidiques, plus précisément un procédé de confection de la partie formant membrane d'analyse, qui ne souffre pas des inconvénients sus-évoqués. Elle vise plus particulièrement à améliorer la méthode décrite dans
Un objectif de la présente invention consiste donc à proposer un procédé de fabrication de membranes d'analyse pour dispositifs microfluidiques dont la mise en oeuvre ne soit pas limitée à la transformation de feuilles en fibre de cellulose ou de nitrocellulose, mais également à des feuilles faites dans d'autres matériaux poreux, ainsi qu'à des feuilles de substrat d'épaisseur relativement importante, c'est-à-dire d'épaisseur au moins égale à de l'ordre de 200 µm, notamment comprise entre 200 µm et 1000 µm.An object of the present invention is therefore to provide a method of manufacturing analysis membranes for microfluidic devices whose implementation is not limited to the processing of sheets of cellulose fiber or nitrocellulose, but also to made sheets. in other porous materials, as well as substrate sheets of relatively large thickness, that is to say with a thickness of at least 200 μm, in particular between 200 μm and 1000 μm. .mu.m.
Un autre objectif de la présente invention est aussi de pouvoir proposer un procédé qui soit compatible avec les contraintes d'une exploitation industrielle d'un dispositif de diagnostic à usage unique, notamment en termes de coût de production et de rentabilité.Another objective of the present invention is also to be able to propose a method that is compatible with the constraints of an industrial operation of a single-use diagnostic device, particularly in terms of production cost and profitability.
La présente invention vise enfin à proposer de nouvelles membranes d'analyse, dont l'originalité se situe non seulement au niveau de leur conception mais également au niveau de leur structure et/ou de la nature des substrats utilisés, ainsi qu'au niveau des performances analytiques susceptibles d'être procurées.Finally, the present invention aims at proposing new analysis membranes whose originality lies not only in their design but also in their structure and / or the nature of the substrates used, as well as in the analytical performance that can be provided.
La présente invention concerne donc, en premier lieu, un procédé de fabrication d'une membrane d'analyse de dispositif microfluidique ; ladite membrane microfluidique étant formée à partir d'une feuille de substrat poreux, dans l'épaisseur de laquelle de la cire solide forme des bordures imperméables délimitant des zones hydrophiles. Ces bordures imperméables décrivent ainsi, à travers ladite feuille de substrat, un motif tracé à la cire.The present invention therefore relates, firstly, to a method of manufacturing a microfluidic device analysis membrane; said microfluidic membrane being formed from a porous substrate sheet, in the thickness of which solid wax forms impermeable edges defining hydrophilic zones. These impermeable edges thus describe, through said substrate sheet, a pattern drawn with wax.
Conformément à l'invention, le procédé comprend les étapes suivantes :
- un motif intermédiaire, façonné à la cire à l'image dudit motif tracé à la cire que formeront les bordures imperméables de la membrane d'analyse, est apposé sur chacune des faces d'une feuille de substrat poreux de sorte que, de part et d'autre de l'épaisseur de ladite feuille de substrat, lesdits motifs intermédiaires, mutuellement symétriques, se trouvent positionnés au moins sensiblement en regard l'un de l'autre ;
- maintenue au moins sensiblement à l'horizontale, ladite feuille de substrat est soumise à un traitement thermique apte à provoquer une fusion au moins partielle de la cire constitutive des motifs intermédiaires apposés sur les faces de ladite feuille de substrat, et à un traitement mécanique apte à comprimer momentanément l'épaisseur de tout ou partie de ladite feuille de substrat ;
- ladite feuille de substrat est soumise à une phase de détente mécanique et thermique, apte à permettre à ladite feuille de substrat de reprendre au moins en partie son épaisseur initiale et à la cire de se resolidifier à l'intérieur de l'épaisseur de ladite feuille de substrat.
- an intermediate pattern waxed in the image of said wax pattern formed by the impermeable edges of the analysis membrane is affixed to each of the faces of a porous substrate sheet so that, on both sides, other than the thickness of said substrate sheet, said intermediate units, mutually symmetrical, are positioned at least substantially opposite one another;
- maintained at least substantially horizontally, said substrate sheet is subjected to a heat treatment capable of causing at least partial melting of the wax constituting the intermediate units affixed to the faces of said substrate sheet, and suitable mechanical treatment temporarily compressing the thickness of all or part of said substrate sheet;
- said substrate sheet is subjected to a mechanical and thermal expansion phase, able to allow said substrate sheet to take up at least part of its initial thickness and the wax to resolidify within the thickness of said sheet of substrate.
Eventuellement, ladite feuille de substrat ainsi transformée est redécoupée aux dimensions finales de la membrane d'analyse souhaitée. Eventuellement, avant ou après cette découpe, la face inférieure est recouverte d'une couche d'étanchéité, par exemple, par pulvérisation ou par application d'une composition de résine ou d'une cire, ou par contre-collage d'un film plastique imperméable, par exemple de type poly(téréphtalate d'éthylène) ou PET. Egalement, de manière intrinsèque, cette couche d'étanchéité peut aussi avoir l'avantage de rigidifier, de renforcer la structure de ladite membrane d'analyse.Optionally, said substrate sheet thus transformed is re-cut to the final dimensions of the desired analysis membrane. Optionally, before or after this cutting, the lower face is covered with a sealing layer, for example, by spraying or by applying a resin composition or a wax, or by laminating an impermeable plastic film, for example of the poly (ethylene terephthalate) or PET type. Also, intrinsically, this sealing layer may also have the advantage of stiffening and strengthening the structure of said analysis membrane.
Dans le présent texte, le terme de « feuille » est utilisé dans un sens très général et large pour désigner une pièce, un morceau de substrat dont l'épaisseur correspond à une valeur insignifiante par rapport à sa surface. Dans ce contexte, ce terme est ici utilisé pour désigner aussi bien une feuille au sens stricte, qu'un ruban ou un disque. De façon relative à cette notion de « feuille », le terme de « film » sera utilisé ci-après pour désigner une pièce, un morceau de matière dont l'épaisseur est significativement inférieure à celle d'une feuille prise au sens de la présente invention.In the present text, the term "sheet" is used in a very general and broad sense to designate a part, a piece of substrate whose thickness corresponds to an insignificant value with respect to its surface. In this context, this term is used here to designate a sheet in the strict sense, a ribbon or a disc. In a manner relative to this notion of "sheet", the term "film" will be used hereinafter to denote a piece, a piece of material whose thickness is significantly less than that of a sheet taken in the sense of the present invention.
Egalement, au sens de la présente invention, le terme de « cires » désigne des substances lipophiles, solides à température ambiante (25°C) et ayant un point de fusion compris entre 45°C et 120°C. Juste au-dessus du point de fusion, les cires sont liquides et ont une viscosité particulièrement basse. Elles peuvent être d'origine animale (par exemple, la cire d'abeille, la cire de Chine, la lanoline...), végétale (par exemple, la cire de riz, de mimosa, la cire de candellila, de carnauba, la cire du Japon, la végétaline...), minérale (par exemple, la paraffine, l'ozocérite...) ou synthétique (par exemple, la stéarine, les polymères éthyléniques, les amines grasses, le polyéthylène glycol ou PEG,...).Also, within the meaning of the present invention, the term "waxes" denotes lipophilic substances, solid at room temperature (25 ° C.) and having a melting point of between 45 ° C. and 120 ° C. Just above the melting point, the waxes are liquid and have a particularly low viscosity. They can be of animal origin (for example, beeswax, Chinese wax, lanolin ...), vegetable (for example, rice wax, mimosa, candellila wax, carnauba, the wax of Japan, the vegetaline ...), mineral (for example, paraffin, ozokerite ...) or synthetic (for example, stearin, ethylene polymers, fatty amines, polyethylene glycol or PEG, ...).
S'agissant de l'étape de traitement thermique du substrat, on prendra soin de ne pas chauffer la cire de façon trop brutale. Une montée en température progressive permettra à la cire déposée sur la face supérieure de la feuille de substrat de fondre lentement et de descendre progressivement dans l'épaisseur du substrat. On évite/limite ainsi la saturation au niveau des couches les plus superficielles du substrat et on minimise, de ce fait, la diffusion latérale/radiale de la cire.Regarding the heat treatment step of the substrate, care should be taken not to heat the wax too brutally. A gradual rise in temperature will allow the wax deposited on the upper surface of the substrate sheet to melt slowly and gradually descend into the thickness of the substrate. This avoids / limits the saturation at the most superficial layers of the substrate and thereby minimizes the lateral / radial diffusion of the wax.
La cire déposée sur la face inférieure de la feuille de substrat imprègne quant à elle progressivement le substrat dans toutes les directions, y compris vers le haut.The wax deposited on the underside of the substrate sheet gradually permeates the substrate in all directions, including upwards.
En comprimant l'épaisseur de la feuille de substrat, on réduit le trajet vertical de la cire. Ce faisant, l'imprégnation en profondeur est favorisée et le temps de migration est raccourci. Moins de liberté est donc laissée à la cire pour pouvoir diffuser dans une direction radiale/latérale ; la finesse du tracé de cire initial s'en trouve affectée de façon moins significative.By compressing the thickness of the substrate sheet, the vertical path of the wax is reduced. In doing so, the deep impregnation is favored and the migration time is shortened. Less freedom is left to the wax to be able to diffuse in one direction radial / lateral; the smoothness of the initial wax pattern is affected less significantly.
Avantageusement et selon l'invention, l'épaisseur de ladite feuille de substrat est comprise entre 200 µm et 1000 µm, préférentiellement entre de l'ordre de 250 µm et de l'ordre de 850 µmAdvantageously and according to the invention, the thickness of said substrate sheet is between 200 μm and 1000 μm, preferably between about 250 μm and about 850 μm.
Selon un mode de mise en oeuvre particulier du procédé selon l'invention, ledit traitement thermique est mis en oeuvre à l'intérieur d'une enceinte chauffante thermo-contrôlée. Quant audit traitement mécanique, celui-ci est mis en oeuvre grâce à des moyens mécaniques aptes à appliquer une pression ou une force de compression suffisante pour comprimer l'épaisseur de ladite feuille de substrat, au moins au niveau des motifs intermédiaires, installés à l'intérieur de ladite enceinte thermo-contrôlée.According to a particular embodiment of the process according to the invention, said heat treatment is carried out inside a thermo-controlled heating chamber. As for said mechanical treatment, it is implemented by mechanical means able to apply a pressure or a compressive force sufficient to compress the thickness of said substrate sheet, at least at the intermediate units, installed at the same time. interior of said thermo-controlled enclosure.
A titre d'exemple, ces moyens mécaniques peuvent consister en une ou plusieurs plaques, ayant une certaine masse, que l'on applique sur la feuille de substrat. Il peut aussi s'agir d'un dispositif formant étau, adapté pour pouvoir maintenir, entre ses mors, la feuille de substrat dans une position au moins sensiblement horizontale, et pour en comprimer l'épaisseur.By way of example, these mechanical means may consist of one or more plates, having a certain mass, which is applied to the substrate sheet. It may also be a vise device, adapted to maintain, between its jaws, the substrate sheet in a position at least substantially horizontal, and to compress the thickness.
Selon une variante de mise en oeuvre du procédé selon l'invention, le traitement thermique et le traitement mécanique sont réalisés simultanément au moyen d'un dispositif de compression chauffant thermo-contrôlé, muni de deux plaques de compression chauffantes horizontales. Il peut s'agir notamment d'une presse à chaud (ou presse chauffante).According to an alternative embodiment of the method according to the invention, the heat treatment and the mechanical treatment are carried out simultaneously by means of a thermo-controlled heating compression device, provided with two horizontal heating compression plates. This may include a hot press (or hot press).
Quel que soit le mode de mise en oeuvre envisagé pour réaliser les traitements thermique et mécanique, on prendra soin d'éviter que la cire déposée sur les faces de la feuille de substrat ne soit en contact direct avec la(les) source(s) de chaleur, ceci essentiellement pour éviter une fusion rapide et brutale de la cire. Lorsqu'un dispositif de compression thermo-régulé est utilisé, une couche de matériau souple et élastique, au moins sensiblement plane, est interposée entre chacune des faces de ladite feuille de substrat et les plaques de compression chauffantes. Ladite couche de matériau souple et élastique fait alors office de tampon thermique et mécanique ; elle permet d'une part de ralentir la transmission de la chaleur au substrat et, d'autre part, de répartir de façon plus homogène la chaleur et la pression sur toute la feuille de substrat. A titre d'élément formant tampon thermique et mécanique, on peut par exemple utiliser une pièce de caoutchouc ou tout autre matériau polymère analogue, une pièce de tissus, une pièce de liège.Whatever the method of implementation envisaged for carrying out the heat and mechanical treatments, care should be taken to avoid that the wax deposited on the faces of the substrate sheet is in direct contact with the source (s). heat, this is essentially to prevent a rapid and sudden melting of the wax. When a thermo-controlled compression device is used, a layer of flexible and elastic material, at least substantially flat, is interposed between each of the faces of said substrate sheet and the heating compression plates. Said layer of flexible and elastic material then serves as a thermal and mechanical buffer; On the one hand, it makes it possible to slow the transmission of heat substrate and, on the other hand, more evenly distribute the heat and pressure over the entire substrate sheet. As a thermal and mechanical buffer element, one can for example use a piece of rubber or any other similar polymeric material, a piece of tissue, a piece of cork.
Avantageusement, on pourra également intercaler une couche de matériau absorbant entre chaque motif intermédiaire, apposé sur une des faces de la feuille de substrat, et ledit tampon thermique et mécanique. Cette couche de matériau absorbant est destinée à capter rapidement la cire fondue restée à la surface du substrat avant que celle-ci ne bave ou ne coule trop abondamment sur les côtés, ce qui pourrait affecter significativement la finesse du tracé de cire.Advantageously, it is also possible to insert a layer of absorbent material between each intermediate pattern, affixed to one of the faces of the substrate sheet, and said thermal and mechanical buffer. This layer of absorbent material is intended to quickly capture the molten wax remaining on the surface of the substrate before it slime or run too much on the sides, which could significantly affect the fineness of the wax pattern.
S'agissant de l'étape de détente thermique et mécanique, aucune précaution particulière à prendre n'est à noter. Le refroidissement peut être effectué de façon passive, en arrêtant le chauffage et/ou en retirant et en éloignant la feuille de substrat de toute source de chaleur. De même, la détente mécanique peut être réalisée simplement en supprimant promptement les contraintes mécaniques appliquées ou en réduisant progressivement leur intensité.Regarding the thermal and mechanical expansion step, no particular precautions to take is noted. Cooling can be done passively, stopping heating and / or removing and moving the substrate sheet away from any heat source. Similarly, mechanical expansion can be achieved simply by quickly removing the mechanical stresses applied or by gradually reducing their intensity.
Egalement, l'ordre chronologique selon lequel on déclenche la détente thermique et la détente mécanique ne revêt pas une grande importance. Avantageusement et selon l'invention, les deux détentes sont déclenchées de façon simultanée ou l'une après l'autre.Also, the chronological order in which thermal expansion and mechanical expansion are triggered is not very important. Advantageously and according to the invention, the two detents are triggered simultaneously or one after the other.
Pour apposer, sur les faces de la feuille de substrat, les motifs intermédiaires façonnés à la cire, toutes les techniques adaptées connues de l'homme du métier peuvent être utilisées.To affix, on the faces of the substrate sheet, the wax-shaped intermediate patterns, any suitable techniques known to those skilled in the art can be used.
A cet effet, l'impression 3D peut être utilisée. En procédant face par face, les motifs intermédiaires sont tracés directement sur la feuille de substrat par dépôt de cire. Sur la face supérieure de la feuille de substrat, le motif intermédiaire reproduit à l'identique le motif final souhaité. Sur la face inférieure, le motif intermédiaire apparait comme une image inversée du motif final souhaité. De part et d'autre de l'épaisseur de la feuille de substrat, les deux motifs intermédiaires, mutuellement symétriques, se trouvent positionnés au moins sensiblement en regard l'un de l'autre.For this purpose, 3D printing can be used. By proceeding face-to-face, the intermediate patterns are traced directly on the substrate sheet by deposition of wax. On the upper face of the substrate sheet, the intermediate pattern reproduces identically the desired final pattern. On the underside, the intermediate pattern appears as an inverted image of the desired final pattern. On either side of the thickness of the substrate sheet, the two intermediate units, mutually symmetrical, are positioned at least substantially opposite one another.
Selon un autre mode de réalisation avantageux, les motifs intermédiaires réalisés à la cire sont apposés sur chacune des faces de la feuille de substrat poreux avec une technique de transfert de cire. A cet effet, les motifs intermédiaires sont d'abord imprimés sur un film de transfert. Sur ce film de transfert imprimé, le motif intermédiaire destiné à revêtir la face inférieure de la feuille de substrat apparait identique au motif final à réaliser, alors que le motif intermédiaire destiné à revêtir la face supérieure de la feuille de substrat, apparait comme une image inversée du premier motif intermédiaire.According to another advantageous embodiment, the waxed intermediate units are affixed to each of the faces of the porous substrate sheet with a wax transfer technique. For this purpose, the intermediate patterns are first printed on a transfer film. On this printed transfer film, the intermediate pattern intended to coat the underside of the substrate sheet appears identical to the final pattern to be produced, whereas the intermediate pattern intended to coat the upper face of the substrate sheet appears as an image. inverted of the first intermediate pattern.
Ces motifs intermédiaires sont ensuite transférés sur chacune des faces de la feuille de substrat, puis dans son épaisseur, en procédant aux traitements thermique et mécanique selon l'invention.These intermediate units are then transferred to each of the faces of the substrate sheet, then in its thickness, by carrying out the thermal and mechanical treatments according to the invention.
En choisissant des films de transfert d'épaisseur relativement faible (par exemple, une épaisseur inférieure à 200 µm), une imprimante à encre solide peut avantageusement être utilisée pour imprimer les motifs intermédiaires. En utilisant une imprimante à encre solide, la quantité (par unité de surface) de cire déposée sur le film de transfert dépend de la qualité d'impression choisie, des encres utilisées -plus précisément de la concentration en cire présente dans ces compositions d'encre-, et de l'intensité de la coloration des motifs à imprimer. En jouant sur ces différents paramètres, un opérateur appréciera le meilleur réglage à appliquer en vue d'obtenir des bordures parfaitement imperméables, qui traversent toute l'épaisseur de la feuille de substrat poreux à transformer/remanier.By choosing transfer films of relatively small thickness (for example, less than 200 μm thick), a solid ink printer can advantageously be used to print the intermediate patterns. By using a solid ink printer, the amount (per unit area) of wax deposited on the transfer film depends on the print quality chosen, the inks used - more precisely the wax concentration present in these compositions. ink-, and the intensity of the coloring of the patterns to be printed. By playing on these different parameters, an operator will appreciate the best setting to be applied in order to obtain perfectly impermeable borders, which cross the entire thickness of the porous substrate sheet to be transformed / remodeled.
En guise de film de transfert utilisable pour la mise en oeuvre du procédé selon l'invention, divers matériaux sont envisageables, dès lors qu'ils sont imprimables. Il peut s'agir notamment d'une feuille plastique imprimable (transparente ou opaque), d'une feuille de papier d'impression, d'une feuille de papier sulfurisée.As transfer film usable for the implementation of the method according to the invention, various materials are possible, since they are printable. This may include a printable plastic sheet (transparent or opaque), a sheet of printing paper, a sheet of parchment paper.
Avantageusement et selon l'invention, les deux motifs intermédiaires, destinés à revêtir les faces supérieure et inférieure de la feuille de substrat sont imprimés sur deux films de transfert distincts. La feuille de substrat est alors placée entre les deux films de transfert, les motifs en cire étant plaqués contre les faces de la feuille de substrat. L'ensemble est maintenu solidaire et est soumis aux traitements thermique et mécanique selon l'invention.Advantageously and according to the invention, the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on two separate transfer films. The substrate sheet is then placed between the two transfer films, the wax patterns being pressed against the faces of the substrate sheet. The assembly is held together and is subjected to heat and mechanical treatments according to the invention.
Selon une variante de mise en oeuvre, les deux motifs intermédiaires destinés à revêtir les faces supérieure et inférieure de la feuille de substrat sont imprimés sur un seul et même film de transfert. Sur ce film de transfert, les deux motifs intermédiaires sont disposés côte à côte, symétriquement par rapport à un axe. Une fois les motifs intermédiaires imprimés et la cire séchée, le film de transfert est plié en deux le long dudit axe de symétrie, la face imprimée tournée vers l'intérieur. Les deux motifs intermédiaires se retrouvent donc superposés. La feuille de substrat est insérée à l'intérieur du film de transfert replié, entre les deux motifs intermédiaires imprimés. L'ensemble est maintenu solidaire et est soumis auxdits traitements thermique et mécanique selon l'invention.According to an alternative embodiment, the two intermediate units intended to coat the upper and lower faces of the substrate sheet are printed on one and the same transfer film. On this transfer film, the two intermediate units are arranged side by side, symmetrically with respect to an axis. Once the intermediate patterns are printed and the wax is dried, the transfer film is folded in half along said axis of symmetry with the printed side turned inward. The two intermediate patterns are thus superimposed. The substrate sheet is inserted inside the folded transfer film between the two printed intermediate patterns. The assembly is held together and is subjected to said thermal and mechanical treatments according to the invention.
Avantageusement et selon l'invention, le film de transfert est une feuille de papier en fibre de cellulose. Légèrement absorbante, ce film permet, lors des traitements thermique et mécanique selon l'invention, de capter au moins une partie de la cire fondue restée à la surface du substrat avant qu'elle ne bave/coule sur les côtés.Advantageously and according to the invention, the transfer film is a paper sheet of cellulose fiber. Slightly absorbent, this film makes it possible, during the thermal and mechanical treatments according to the invention, to capture at least a portion of the molten wax remaining on the surface of the substrate before it slinks / flows on the sides.
Le procédé selon l'invention peut être mis en oeuvre à partir d'une large gamme de feuilles de substrat poreux, notamment de composition fibreuse choisie parmi : la fibre de cellulose, de nitrocellulose, de coton, de linter, de verre, de soie, de viscose, de polypropylène, de polyester, de polyamide (Nylon®), de poly(acide lactique) ou PLA. Lesdites fibres peuvent éventuellement être fonctionnalisées et/ou chargées et/ou dopées d'additifs (par exemple, talc, diatomite...).The method according to the invention can be implemented from a wide range of porous substrate sheets, in particular from a fibrous composition chosen from: cellulose, nitrocellulose, cotton, linter, glass, silk fiber , viscose, polypropylene, polyester, polyamide (Nylon ® ), poly (lactic acid) or PLA. Said fibers may optionally be functionalized and / or loaded and / or doped with additives (for example, talc, diatomite, etc.).
Avantageusement, le procédé selon l'invention est mis en oeuvre à partir d'une feuille de substrat poreux de même composition qu'un média filtrant réalisé en fibre de cellulose, de nitrocellulose, de coton, de linter, de soie, de viscose, de polypropylène, de polyester, préférentiellement en fibre de verre.Advantageously, the process according to the invention is carried out starting from a porous substrate sheet of the same composition as a filtering medium made of cellulose fiber, nitrocellulose, cotton, linter, silk, viscose, polypropylene, polyester, preferably fiberglass.
Au sens de la présente invention et de façon générale, le terme de « média filtrant » désigne un matériau filtrant classiquement utilisé dans les laboratoires et dans l'industrie à des fins d'analyse de liquides, et plus spécifiquement à des fins de séparation et de purification. En fonction de leur épaisseur et de leur format, les qualificatifs de membrane, papier, tampon ou disque, leur sont parfois attribués. De même, on entend par « média filtrant en fibre de verre », un matériau filtrant réalisé à base de microfibres de verre, éventuellement borosilicatés, avec ou sans liant. A titre d'exemples de médias filtrants en fibre de verre actuellement disponibles dans le commerce, on peut notamment citer :
- les filtres WHATMAN™ (GE Healthcare Life Sciences, États-Unis), notamment les papiers MF1, LF1, VF1, VF2 et Fusion 5, destinés à la filtration du sang total et à la séparation cellules/plasma, et le filtre GF/C, destiné à la séparation de substances solides en suspension dans un fluide ;
- les filtres de la société SARTORIUS A.G. (Allemagne), notamment ceux destinés au traitement et à l'analyse de fluide.
- WHATMAN ™ filters (GE Healthcare Life Sciences, USA), including MF1, LF1, VF1, VF2 and Fusion 5, for whole blood filtration and cell / plasma separation, and the GF / C filter for separating suspended solids in a fluid;
- the filters of the company SARTORIUS AG (Germany), in particular those intended for the treatment and analysis of fluid.
Avantageusement et selon l'invention, ledit procédé de fabrication d'une membrane d'analyse de dispositif microfluidique est mis en oeuvre à partir d'une feuille de substrat poreux en fibre de verre. Avantageusement et selon l'invention, ladite feuille de substrat reprend la composition d'un média filtrant en fibre de verre.Advantageously and according to the invention, said method of manufacturing a microfluidic device analysis membrane is implemented from a porous fiberglass substrate sheet. Advantageously and according to the invention, said substrate sheet contains the composition of a fiberglass filter medium.
Les médias filtrants en fibre de verre se caractérisent par une très grande fragilité à la déchirure et l'effritement. Cette grande fragilité structurelle les rend incompatibles à toute impression directe au moyen des imprimantes à encre solide actuellement proposées dans le commerce. Les contraintes mécaniques imposées par ces dernières leur sont en effet considérablement dénaturantes.Fiberglass filtering media are characterized by a great fragility in tearing and crumbling. This great structural fragility makes them incompatible with any direct printing by means of solid ink printers currently on the market. The mechanical constraints imposed by them are indeed considerably denaturing.
Ainsi, outre le fait d'avoir envisagé d'utiliser un média filtrant en fibre de verre comme substrat poreux pour la confection de membranes d'analyse de/ou pour dispositifs microfluidiques, il est du mérite des inventeurs d'être parvenus à mettre au point un procédé apte à intégrer dans l'épaisseur de ce matériau si particulier, de la cire pour y former in fine des bordures imperméables délimitant des zones dites hydrophiles. Qui plus est, outre la simplicité et le faible coût de sa mise en oeuvre, le procédé proposé permet la réalisation, dans l'épaisseur des feuilles de substrat d'épaisseur relativement importante (comprise entre de 200 µm et 1000 µm), de bordures de cire parfaitement imperméables aux liquides et dont la finesse (épaisseur latérale) peut atteindre de l'ordre de 800 µm.Thus, in addition to having considered the use of a fiberglass filtering medium as a porous substrate for making analysis membranes of / or for microfluidic devices, it is the merit of the inventors to have succeeded in point a process capable of integrating into the thickness of this particular material, wax to form in fine impervious edges defining so-called hydrophilic zones. Moreover, in addition to the simplicity and low cost of its implementation, the proposed method allows the production, in the thickness of the relatively thick substrate sheets (between 200 microns and 1000 microns), curbs waxes perfectly impermeable to liquids and whose fineness (lateral thickness) can reach of the order of 800 microns.
La présente invention concerne, en deuxième lieu, une membrane d'analyse de dispositif microfluidique, susceptible d'être fabriquée par un procédé selon l'invention. Ladite membrane d'analyse présente une épaisseur de laquelle de la cire solide forme des bordures imperméables (en l'occurrence, imperméables aux liquides) délimitant des zones hydrophiles. Selon l'invention, l'épaisseur de la membrane d'analyse est comprise entre 200 µm et 1000 µm, préférentiellement entre de l'ordre de 250 µm et de l'ordre de 850 µm.The present invention relates, secondly, a microfluidic device analysis membrane, capable of being manufactured by a method according to the invention. The said test membrane has a thickness of which solid wax forms impermeable (in this case, liquid impermeable) borders delimiting hydrophilic zones. According to the invention, the thickness of the analysis membrane is between 200 μm and 1000 μm, preferably between about 250 μm and about 850 μm.
Eventuellement, ladite membrane d'analyse comprend une face (en l'occurrence, une face dite face inférieure) recouverte d'une couche d'étanchéité. Celle-ci peut être obtenue par pulvérisation ou par application d'une composition de résine ou d'une cire, ou par contre-collage d'un film plastique, par exemple de type PET.Optionally, said analysis membrane comprises a face (in this case, a so-called lower face) covered with a sealing layer. This can be obtained by spraying or by applying a resin composition or a wax, or by laminating a plastic film, for example of the PET type.
Avantageusement et selon l'invention, la cire constitutive desdites bordures imperméables est choisie parmi une cire animale (par exemple, la cire d'abeille, la cire de Chine, la lanoline...), végétale (par exemple, la cire de riz, de mimosa, la cire de candellila, de carnauba, la cire du Japon, la végétaline...), minérale (par exemple, la paraffine, l'ozocérite...) et synthétique (par exemple, la stéarine, les polymères éthyléniques, les amines grasses, le polyéthylène de glycol ou PEG...).Advantageously and according to the invention, the constitutive wax of said impermeable edges is chosen from an animal wax (for example, beeswax, Chinese wax, lanolin, etc.) and vegetable (for example, rice wax). , mimosa, candellila wax, carnauba wax, Japan wax, vegetaline ...), mineral (for example, paraffin, ozokerite ...) and synthetic (for example, stearin, polymers ethylenic, fatty amines, polyethylene glycol or PEG ...).
Avantageusement et selon l'invention, la membrane d'analyse est en fibre de cellulose, en fibre de nitrocellulose ou en fibre de verre, en coton, en linter, en ouate de cellulose, en fibre de soie, de viscose, de polypropylène, de polyester. Elle peut éventuellement être fonctionnalisée et/ou chargée et/ou dopée d'additifs (par exemple, talc, diatomite...).Advantageously and according to the invention, the analysis membrane is made of cellulose fiber, nitrocellulose fiber or fiberglass, cotton, linter, cellulose wadding, silk fiber, viscose, polypropylene, of polyester. It may optionally be functionalized and / or charged and / or doped with additives (for example, talc, diatomite, etc.).
Avantageusement et selon l'invention, la membrane d'analyse est réalisée dans un substrat poreux dont la composition reprend celle d'un média filtrant en fibre de cellulose, de nitrocellulose, de coton, de linter, de verre, de soie, de viscose, de polypropylène, de polyester, de polyamide ou de PLA.Advantageously and according to the invention, the analysis membrane is made in a porous substrate whose composition resumes that of a filtering medium of cellulose fiber, nitrocellulose, cotton, linter, glass, silk, viscose , polypropylene, polyester, polyamide or PLA.
Selon un mode de réalisation préférentiel, la composition de la membrane d'analyse est en fibre de verre. Elle reprend avantageusement la composition d'un média filtrant tel que notamment :
- les filtres WHATMAN™ de la compagnie GE HEALTHCARE LIFE SCIENCE, (États-Unis), en particulier les filtres MF1, LF1, VF1 et VF2, qui sont ordinairement destinés à la filtration du sang total et à la séparation cellules/plasma, et du filtre GF/C, et/ou à la séparation de substances solides en suspension dans un fluide ;
- les filtres de la société SARTORIUS A.G. (Allemagne), notamment ceux destinés au traitement et à l'analyse de fluides.
- WHATMAN ™ filters from GE HEALTHCARE LIFE SCIENCE, (USA), particularly the MF1, LF1, VF1 and VF2 filters, which are typically intended for whole blood filtration and cell / plasma separation, and GF / C filter, and / or separation of suspended solids in a fluid;
- the filters of the company SARTORIUS AG (Germany), in particular those intended for the treatment and analysis of fluids.
Avantageusement et selon ce mode de réalisation préférentiel, la membrane d'analyse a une épaisseur comprise entre de 200 µm et 1 000 µm, typiquement comprise entre de l'ordre de 250 µm et de l'ordre de 850 µm.Advantageously and according to this preferred embodiment, the analysis membrane has a thickness of between 200 microns and 1000 microns, typically between about 250 microns and about 850 microns.
Les membranes d'analyse réalisées en fibre de verre selon l'invention permettent une analyse du plasma directement à partir du sang total. Le sang déposé à la surface de la membrane est fractionné ; les cellules et le plasma sont séparés. Les cellules restent fixées dans la zone de dépôt alors que le plasma va diffuser à travers la membrane, en progressant le long des canaux hydrophiles par diffusion latérale.The analysis membranes made of fiberglass according to the invention allow an analysis of the plasma directly from the whole blood. The blood deposited on the surface of the membrane is fractionated; cells and plasma are separated. The cells remain fixed in the deposition zone while the plasma will diffuse through the membrane, progressing along the hydrophilic channels by lateral diffusion.
Dans les dispositifs microfluidiques actuellement disponibles dans le commerce, les membranes d'analyse sont classiquement réalisées en fibre de cellulose ou de nitrocellulose. A elles seules, ces membranes d'analyse sont incapables de séparer la fraction plasmatique de la fraction cellulaire. Le sang total déposé sur de telles membranes d'analyse coagule rapidement ; après le dépôt de l'échantillon de sang sur ces supports, les cellules bloquent le plasma et empêche sa diffusion latérale à travers les canaux hydrophiles. Ainsi, pour les analyses sanguines, les µPAD jusqu'à présent proposés comprennent un assemblage d'une membrane d'analyse avec un système de filtration (par exemple, un média filtrant en fibre de verre) apte à mettre en oeuvre un fractionnement du sang ; une fois séparé des cellules qui restent bloquées au niveau du système de filtration, le plasma diffuse à travers la membrane d'analyse où ses composants sont alors analysés. Un exemple de µPAD comprenant une tel assemblage entre une membrane d'analyse et un média filtrant est décrit par
Pour les modèles de µPAD ne disposant pas d'un tel de système de filtration, ceux-ci doivent être utilisés avec du plasma préalablement purifié.For μPAD models that do not have such a filtration system, they must be used with previously purified plasma.
Dans un mode de réalisation particulier, outre des bordures imperméables tracées à la cire et délimitant des zones hydrophiles, ladite membrane d'analyse comprend également dans son épaisseur des incrustations de cire, présentes au niveau d'au moins une zone hydrophile. Ces incrustations de cire ne forment pas des barrières imperméables au passage des liquides. Positionnées au niveau de diverses zones hydrophiles, elles ont vocation à perturber et/ou à ralentir localement le flux de liquide et/ou à créer des zones réservoirs et/ou de réaction bien déterminées. Elles peuvent aussi définir les contours de zones sans cire, donc très hydrophiles, qui pourront faire usage de réservoirs ou de zones réactionnelles bien délimitées.In a particular embodiment, in addition to impermeable edges drawn with wax and delimiting hydrophilic zones, said analysis membrane also comprises, in its thickness, encrustations of wax, present at the level of at least one hydrophilic zone. These wax encrustations do not form impermeable barriers to passage of liquids. Positioned at various hydrophilic zones, they are intended to disrupt and / or slow down the flow of liquid locally and / or to create well-defined reservoir and / or reaction zones. They can also define the contours of wax-free zones, thus very hydrophilic, which can make use of reservoirs or well-defined reaction zones.
Ces incrustations de cire peuvent être réalisées de multiples façons. Une première méthode consiste à reprendre le procédé précédemment décrit pour établir les bordures imperméable de la membrane d'analyse. On obtient alors des incrustations ponctuelles et localisées de cire qui s'étendent à travers toute l'épaisseur de ladite feuille. Elles forment ainsi des obstacles que les liquides doivent contourner par les côtés pour pouvoir progresser à travers la membrane d'analyse.These wax encrustations can be made in many ways. A first method consists of taking the previously described method to establish the impermeable edges of the analysis membrane. Localized and localized incrustations of wax are then obtained which extend through the entire thickness of said sheet. They thus form obstacles which the liquids must circumvent by the sides to be able to progress through the membrane of analysis.
Une autre méthode consiste à répéter le procédé selon l'invention mais en le modifiant quelque peu pour ne disperser dans l'épaisseur du substrat poreux qu'une quantité faible de cire, insuffisante pour pouvoir bloquer complètement la migration des liquides. A cet effet, on peut par exemple, utiliser une encre solide faiblement concentrée en cire et/ou n'apposer l'encre solide que sur une des deux faces de la feuille de substrat et/ou imprimer le film de transfert avec une couche de couleur pâle, faiblement intense.Another method is to repeat the process according to the invention but by modifying it somewhat to disperse in the thickness of the porous substrate a small amount of wax, insufficient to completely block the migration of liquids. For this purpose, it is possible, for example, to use a solid ink having a low concentration of wax and / or to affix the solid ink only to one of the two faces of the substrate sheet and / or to print the transfer film with a layer of pale color, weakly intense.
Un résultat similaire peut être obtenu en reprenant le procédé selon l'invention, mais cette fois-ci sans comprimer l'épaisseur de la feuille de substrat poreux.A similar result can be obtained by repeating the process according to the invention, but this time without compressing the thickness of the porous substrate sheet.
Par rapport à ce dernier mode de réalisation, les modes précédemment évoqués ont l'avantage de permettre la réalisation des incrustations de cire de façon concomitante avec celles des bordures imperméables.Compared to this last embodiment, the previously mentioned modes have the advantage of allowing the realization of wax encrustations concomitantly with those of impermeable edges.
La présente invention s'étend également à un dispositif microfluidique comprenant au moins une membrane d'analyse selon l'invention.The present invention also extends to a microfluidic device comprising at least one analysis membrane according to the invention.
La présente invention concerne également un procédé de fabrication d'une membrane d'analyse de dispositif microfluidique, une membrane d'analyse de dispositif microfluidique ainsi qu'un dispositif microfluidique caractérisés, en combinaison, par tout ou partie des caractéristiques ci-dessus ou ci-après.The present invention also relates to a method for manufacturing a microfluidic device analysis membrane, a microfluidic device analysis membrane and a microfluidic device characterized, in combination, by all or some of the features above or above. -after.
D'autres buts, caractéristiques et avantages de l'invention apparaitront à la lecture des exemples suivants qui se réfèrent aux figures annexées et dans lesquelles :
- la
Figure 1 est une représentation schématisée d'un mode particulier de mise en oeuvre d'un procédé de fabrication d'une membrane d'analyse de dispositif microfluidique conforme à la présente invention ; - la
Figure 2 présente une photographie d'une membrane WHATMAN™ MF1 à la sortie d'une imprimante à encre solide, après une tentative d'impression directe ; - la
Figure 3 expose en (A), une représentation schématique d'un film de transfert sur lequel est imprimé un exemple de motifs intermédiaires et en (B), une photographie d'une membrane d'analyse obtenue à partir de ce film de transfert ; - la
Figure 4 présente deux photographies de membranes d'analyse pour dispositifs microfluidiques ; la photographie (A) correspond à une membrane d'analyse obtenue par mise en oeuvre d'un procédé selon l'invention ; la photographie (B) correspond à une membrane d'analyse obtenue avec le même procédé mais en omettant le traitement mécanique ; - la
Figure 5 est un graphique mettant en évidence une relation linéaire entre l'épaisseur d'un trait de cire imprimé sur un film de transfert et l'épaisseur de ce même trait après transfert dans l'épaisseur d'une feuille de substrat ; - la
Figure 6 expose des photographies permettant d'apprécier l'imperméabilité des bordures de cire façonnées avec un procédé selon l'invention, en fonction de leur épaisseur ; - la
Figure 7 présente deux photographies prises en microscopie ; la première (A) montre une bordure imprimée à la cire sur un film de transfert en fibre de cellulose, la seconde (B) montre cette même bordure après transfert vers une membrane WHATMAN™ MF1 ; - La
Figure 8 montre des photographies illustrant l'imperméabilité de bordures d'épaisseur de l'ordre de 800 µm, formées sur des substrats de différentes compositions et de différentes épaisseurs, par mise en oeuvre d'un procédé selon l'invention ; - les
Figure 9, Figure 10A et Figure 10B sont des photographies illustrant la mise en application d'une membrane d'analyse selon l'invention à des fins d'immunodétection ; et - la
Figure 11 est une photographie d'une membrane d'analyse selon l'invention façonnée pour le dépistage du virus de la dengue, par une codétection des protéines NS1 et DomIII.
- the
Figure 1 is a schematic representation of a particular embodiment of a method of manufacturing a microfluidic device analysis membrane according to the present invention; - the
Figure 2 presents a photograph of a WHATMAN ™ MF1 membrane at the exit of a solid ink printer, after a direct print attempt; - the
Figure 3 discloses in (A), a schematic representation of a transfer film on which is printed an example of intermediate units and in (B), a photograph of an analysis membrane obtained from this transfer film; - the
Figure 4 presents two photographs of analysis membranes for microfluidic devices; the photograph (A) corresponds to an analysis membrane obtained by implementing a method according to the invention; the photograph (B) corresponds to an analysis membrane obtained with the same process but omitting the mechanical treatment; - the
Figure 5 is a graph showing a linear relationship between the thickness of a wax line printed on a transfer film and the thickness of this same line after transfer in the thickness of a substrate sheet; - the
Figure 6 discloses photographs to appreciate the impermeability wax edging shaped with a method according to the invention, depending on their thickness; - the
Figure 7 presents two photographs taken in microscopy; the first (A) shows a wax-printed border on a cellulose fiber transfer film, the second (B) shows the same border after transfer to a WHATMAN ™ MF1 membrane; - The
Figure 8 shows photographs illustrating the impermeability of edges of thickness of the order of 800 microns, formed on substrates of different compositions and thicknesses, by implementing a method according to the invention; - the
Figure 9, Figure 10A and Figure 10B are photographs illustrating the application of an analysis membrane according to the invention for immunodetection purposes; and - the
Figure 11 is a photograph of an analysis membrane according to the invention shaped for the detection of the dengue virus, by a co-detection of the NS1 and DomIII proteins.
La
Dans une première étape, on crée, par informatique et au moyen d'un logiciel de dessin, une image 12'a correspondant au motif à intégrer dans l'épaisseur de la feuille de substrat 11. Cette première image 12'a est dupliquée en une image symétrique 12'b.In a first step, an image 12'a corresponding to the pattern to be integrated in the thickness of the
Au moyen d'une imprimante à encre solide, les deux images 12'a et 12'b sont imprimées sur un film de transfert 20, de façon à former deux motifs intermédiaires 12a et 12b disposés symétriquement par rapport à un axe. Cet axe de symétrie S est également imprimé sur le film de transfert 20, en guise de repère visuel.By means of a solid ink printer, the two images 12'a and 12'b are printed on a
Pour ce faire, l'impression est réalisée avec une imprimante de type XEROX® ColorQube™, alimentée avec des encres solides de références XEROX® 108R00931 (couleur cyan), 108R00932 (couleur magenta), 108R00933 (couleur jaune) et 108R00934/108R00935 (couleur noire). Le film de transfert 20 est une feuille de papier bureautique ordinaire. Les contours du motif sont imprimés en qualité/résolution photo à l'encre noire.To do this, the printing is carried out with a XEROX ® ColorQube ™ type printer, supplied with solid inks of reference XEROX ® 108R00931 (cyan color), 108R00932 (magenta color), 108R00933 (yellow color) and 108R00934 / 108R00935 ( black color). The
Une fois cette impression réalisée, le film de transfert 20 est plié en deux en suivant l'axe de symétrie S, les motifs intermédiaires 12a et 12b tournés vers l'intérieur. Ces derniers se retrouvent ainsi superposés l'un sur l'autre. Une feuille de substrat 11 est glissée à l'intérieur du film de transfert 20 replié, intercalé entre les deux motifs intermédiaires 12a et 12b.Once this impression is made, the
La feuille de substrat 11, prise en sandwich entre les deux volets du film de transfert 20, est alors mise sous presse entre deux plaques de compression chauffantes, horizontales et deux pièces en caoutchouc 15 formant tampon thermique et mécanique.The
L'ensemble est soumis à une pression de l'ordre de 1 kg/cm2 et à une température de 120°C, pendant environ 3 minutes. Lors de ce processus, la cire préalablement imprimée sur le film de transfert 20 est transférée sur les deux faces de la feuille de substrat 11, puis en imprègne l'épaisseur.The assembly is subjected to a pressure of the order of 1 kg / cm 2 and at a temperature of 120 ° C for about 3 minutes. During this process, the wax previously printed on the
Le procédé décrit ici s'applique à de nombreux substrats poreux, tels que par exemple des substrats réalisés en fibre de cellulose, de nitrocellulose, de coton, de linter, de verre, de soie, de viscose, de polypropylène, de polyester, de polyamide, de PLA. Ce procédé a été mis au point essentiellement sur des substrats en fibre de verre et plus particulièrement sur des médias de filtration de marque WHATMAN™ (GE HEALTHCARE LIFE SCIENCE, États-Unis), d'épaisseurs variables, notamment :
- filtre MF1, d'une épaisseur de 367 µm
- filtre Fusion 5, d'une épaisseur de 370 µm
- filtre VF2, d'une épaisseur de 785 µm.
- MF1 filter, with a thickness of 367 μm
- Fusion 5 filter, 370 μm thick
- VF2 filter with a thickness of 785 μm.
Ces médias de filtration habituellement utilisés pour la filtration d'échantillons de sang total, en vue d'une séparation rapide entre fraction cellulaire / fraction liquide, sont extrêmement fragiles à la déchirure et doivent être manipulés avec précaution et délicatesse.These filtration media usually used for the filtration of whole blood samples, for a rapid separation between cellular fraction / liquid fraction, are extremely fragile to the tear and must be handled with care and delicacy.
Compte tenu de cette grande fragilité, il n'est pas possible de procéder à une impression des motifs intermédiaires directement sur ce type de matériau au moyen d'une imprimante à encre solide. La
La
Les motifs intermédiaires 12a et 12b, de forme générale rectiligne, présentent une partie supérieure évasée et une partie basse plus étroite à extrémité ouverte. Leurs contours sont imprimés à l'encre noire (référence XEROX® 108R00934), en qualité photo. Cette encre solide renferme une concentration en cire qui s'est avérée suffisante pour permettre la réalisation et l'obtention de bordures imperméables dans l'épaisseur de feuilles de substrat relativement épaisses, y compris des membranes WHATMAN™ VF2 de 785 µm d'épaisseur.The
La partie supérieure des motifs intermédiaires 12a et 12b est évasée et est destinée à former une zone hydrophile, en l'occurrence une zone de dépôt 13a apte à recevoir un échantillon liquide à analyser. Une fois déposé dans la zone de dépôt 13a, l'échantillon liquide va pouvoir migrer par capillarité en direction des autres zones hydrophiles de la membrane d'analyse, à savoir vers la partie basse du motif. Plus étroite, elle forme un canal à l'intérieur duquel sont ménagées quatre zones d'intérêt 13b, 13c, 13d et 13e. La position de chacune de ces zones d'intérêt est spécifiquement repérée par les éléments de marquage 14b, 14c, 14d et 14e, également tracée à l'encre solide colorée.The upper part of the
Du fait de la grande hydrophilie qui caractérise les médias filtrant en fibre de verre, il peut être avantageux de pouvoir ralentir le flux des liquides et/ou modifier leur profil d'écoulement, notamment au niveau des zones d'intérêt 13b, 13c, 13d et 13e. Pour ce faire, sur la partie basse des motifs intermédiaires 12a et 12b, autours des régions destinées à former les zones d'intérêt 13b, 13c, 13d et 13e, une couche de couleur faiblement chargée en cire 14 recouvre la surface du film de transfert 20. Contrairement aux contours des motifs intermédiaires 12a et 12b qui sont imprimés à l'encre solide noire, la couche faiblement chargée en cire 14 est obtenue en imprimant, à l'encre solide, une couche de couleur pâle (faiblement intense). L'intensité de la couleur de cette couche est proportionnelle à la quantité de cire déposée sur le film de transfert ; cette intensité de couleur est choisie pour que, une fois dans l'épaisseur de la feuille de substrat, la quantité de cire ainsi transférée soit insuffisante pour bloquer complètement la migration des liquides par capillarité.Due to the great hydrophilicity which characterizes fiberglass filtering media, it may be advantageous to be able to slow down the flow of liquids and / or modify their flow profile, particularly at the level of the zones of
Le dépôt d'une couche de couleur jaune pâle sur le film de transfert a permis de réduire et moduler assez précisément les vitesses d'écoulement des liquides au sein de la membrane d'analyse.The deposition of a pale yellow layer on the transfer film made it possible to reduce and modulate quite accurately the flow velocities of the liquids within the analysis membrane.
Les zones d'intérêt 13b, 13c, 13d et 13e n'ont pas été modifiées par la cire. Elles conservent la porosité et l'hydrophilie initiales de la feuille de substrat utilisée, et pourront être fonctionnalisées ultérieurement.Areas of
Une fois les cires transférées dans l'épaisseur de la feuille de substrat, les différentes zones plus ou moins hydrophiles vont pouvoir être fonctionnalisées et/ou chargées en réactifs en fonction des analyses et tests souhaités.Once the waxes are transferred into the thickness of the substrate sheet, the various more or less hydrophilic zones will be able to be functionalized and / or loaded with reagents according to the desired analyzes and tests.
La
La membrane d'analyse de la photographie (A) a été fabriquée en mettant en oeuvre un procédé conforme à l'invention et tel qu'illustré à la
Les deux membranes d'analyse sont chargées d'une solution colorée. A la différence de la membrane d'analyse de la photographie (A), avec celle de la photographie (B), des fuites importantes de la solution colorée sont constatées au niveau de la zone de dépôt. La quantité de cire transférée est nettement plus faible et ne permet pas d'obtenir motif aux parois imperméables.The two analysis membranes are loaded with a colored solution. Unlike the analysis membrane of the photograph (A), with that of the photograph (B), large leaks of the colored solution are observed at the level of the deposition zone. The amount of wax transferred is significantly lower and does not allow to obtain pattern impermeable walls.
- a) Des motifs circulaires avec des épaisseurs de trait variables sont imprimés sur un film de transfert en fibre de cellulose (impression à l'encre solide noir, en qualité photo et au moyen d'une imprimante de type XEROX® ColorQube™), puis transférés sur membrane WHATMAN™ MF1 (367 µm d'épaisseur), par traitements thermique et mécanique selon l'invention, durant 5 minutes à 120°C sous une pression équivalente à 0,5-2 kg.cm-2. Les épaisseurs des traits de cire avant et après transfert sont mesurées sous microscope.a) Circular patterns with variable line widths are printed on a cellulose fiber transfer film (black solid ink printing, photo quality and XEROX ® ColorQube ™ type printer), then transferred on WHATMAN ™ MF1 membrane (367 microns thick), by thermal and mechanical treatments according to the invention, for 5 minutes at 120 ° C. under a pressure equivalent to 0.5-2 kg.cm -2 . The thicknesses of the wax lines before and after transfer are measured under a microscope.
Les résultats obtenus sont présentés à la
- b) Une solution colorée est ensuite déposée au centre de chacun des motifs réalisés pour évaluer l'étanchéité des bordures crées. La
Figure 6 et laFigure 7 présentent les résultats obtenus, sous la forme de photographies.
- b) A colored solution is then deposited in the center of each of the patterns made to evaluate the tightness of the edges created. The
Figure 6 and theFigure 7 present the results obtained in the form of photographs.
L'imperméabilité aux liquides est obtenue pour une épaisseur de cire imprimée au minimum égale à 260 µm, donnant après transfert une barrière de cire de l'ordre de 830 µm En deçà de cette épaisseur, le colorant n'est plus contenu dans le motif circulaire.The impermeability to liquids is obtained for a wax thickness printed at least equal to 260 μm, giving after transfer a wax barrier of the order of 830 μm. Below this thickness, the colorant is no longer contained in the pattern. circular.
Des substrats, d'épaisseur et de nature variables, ont été testés. Il s'agit en l'occurrence des médias filtrants commercialisés par la compagnie GE HEALTHCARE LIFE SCIENCE (États-Unis), sous la marque de fabrique WHATMAN™ : les filtres MF1, Fusion 5 et VF2, respectivement de 367 µm, 370 µm et 785 µm d'épaisseur.Substrates of varying thickness and nature were tested. These are filter media marketed by the company GE HEALTHCARE LIFE SCIENCE (United States), under the trademark WHATMAN ™: MF1 filters, Fusion 5 and VF2, respectively 367 microns, 370 microns and 785 μm thick.
Les transferts, par traitements thermique et mécanique selon l'invention, ont lieu durant 3 minutes à 120°C et sous une pression équivalente à environ 1 kg/cm2. L'étanchéité des motifs est vérifiée à l'aide d'un dépôt d'une solution colorée dans la partie évasée du motif (zone de dépôt).Transfers, by heat and mechanical treatments according to the invention, take place for 3 minutes at 120 ° C. and under a pressure equivalent to about 1 kg / cm 2 . The tightness of the patterns is checked by means of a deposit of a colored solution in the flared portion of the pattern (deposition zone).
Les résultats obtenus sont présentés à la
Selon un premier mode de mise en application de la membrane d'analyse 10 précédemment décrite, celle-ci a été fonctionnalisée pour le dépistage de l'hépatite B, par immunodétection de l'antigène HBs contenu dans le sang.According to a first embodiment of the
Dans ce contexte, des fonctions particulières ont été affectées aux zones d'intérêt 13b, 13c, 13d et 13e.In this context, particular functions have been assigned to areas of
La zone 13d est fonctionnalisée au moyen d'anticorps monoclonaux anti-HBs, spécifiques de la réaction; la zone 13d forme le «spot test ». La zone 13e est fonctionnalisée au moyen d'anticorps monoclonaux anti-alcaline phosphatase, spécifiques du conjugué de détection ; la zone 13e forme le «spot contrôle positif». La zone 13c est fonctionnalisée au moyen d'anticorps non-spécifiques de la réaction (par exemple, des anticorps anti-rat) ; la zone 13c forme le « spot contrôle négatif ».The zone 13d is functionalized by means of monoclonal antibodies anti-HBs, specific for the reaction; zone 13d forms the "spot test". The 13th zone is functionalized by means of anti-alkaline phosphatase monoclonal antibodies, specific for the detection conjugate; the 13th zone forms the "positive control spot". The
La fonctionnalisation de ces différentes zones par les anticorps est réalisée par adsorption passive des anticorps sur les fibres constitutives du substrat.The functionalization of these different zones by the antibodies is carried out by passive adsorption of the antibodies on the constituent fibers of the substrate.
Au-dessus de ces trois spots, la zone 13b est dédiée au stockage de la seconde partie du complexe conjugué (anticorps monoclonaux anti-HBs marqués par la biotine) ; la zone 13b forme le « spot Ac anti-HBs-biot ».Above these three spots,
Eventuellement, la zone de dépôt 13a peut également servir au stockage du conjugué de la réaction immuno-enzymatique (streptavidine-alcaline phosphatase ou STRE-PAL) sous forme séchée. Ce conjugué sera resolubilisé par la phase liquide de l'échantillon à analyser.Optionally, the
Selon un deuxième mode de mise en application de la membrane d'analyse 10 précédemment décrite, celle-ci a été fonctionnalisée pour la détection, dans le sang et le plasma, de deux protéines du virus de la dengue : la protéine NS1 et le domaine III de la protéine d'enveloppe du virus (DomIII).According to a second mode of application of the
Pour la détection de la protéine NS1, les dépôts suivants ont été réalisés :
zone 13e (zone contrôle anti-PAL) : 0,35 µL d'un anticorps anti-phosphatase alcaline 1 mg/mL en PBS,zone 13c (zone de test) : 0,35 µL d'un anticorps anti-NS1 à 1 mg/mL en PBS,zone 13b : 1 µL d'une solution d'anticorps anti-NS1 marqués à la phosphatase alcaline à la concentration de 50 µg/mL en PBS-BSA 0,5 %,- zone 13d (zone de contrôle négatif) : 0,35 µL de PBS-
BSA 0,5 %.
-
zone 13e (anti-ALP control zone): 0.35 μL of ananti-alkaline phosphatase antibody 1 mg / mL in PBS, -
zone 13c (test zone): 0.35 μl of an anti-NS1 antibody at 1 mg / ml in PBS, -
zone 13b: 1 μl of a solution of anti-NS1 antibodies labeled with alkaline phosphatase at the concentration of 50 μg / ml in PBS-BSA 0.5%, - zone 13d (negative control zone): 0.35 μL of PBS-BSA 0.5%.
Pour tester cette membrane d'analyse, 20-25 µL de plasma additionnés de 1 µg de protéine NS1 sont déposés dans la zone réservoir 13a. Une fois que la totalité de l'échantillon a pénétré et migré à l'intérieur du pad, 10µL de BCIP/NTP (bromo-4-chloro-3-indol phosphate/4-nitroblue tetrazolium chloride) sont rajoutés dans la zone 13b. Très rapidement, la zone contrôle anti-PAL et la zone de test deviennent positives, la zone de contrôle négatif restant incolore (cf.
De façon similaire, la membrane d'analyse 10 précédemment décrite, a été fonctionnalisée pour la détection de la protéine de l'enveloppe du virus de la dengue, en utilisant des anticorps de capture et de détection dirigés contre DomIII. La membrane d'analyse correspondante a été testée. Une détection positive de DomIII et une détection négative sont illustrées par les
Enfin, la codétection des protéines NS1 et DomIII a également été mise en oeuvre avec succès au moyen d'une membrane d'analyse (multiplexage) dont la structure et la conception sont très similaires à celles des membranes d'analyse 10 précédentes. La différence repose sur la présence d'une zone de réaction supplémentaire au niveau de l'espace à diffusion ralentie. Une photographie de cette nouvelle membrane d'analyse selon l'invention est présentée sur la
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characterized in that it comprises the following steps:
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